Project Summary/Abstract Atherosclerotic and high-risk plaques including both rupture and erosion are predominantly localized to vessel wall regions with non-laminar disturbed blood flow (d-flow) and laminar high flow (h-flow). However, the molecular mechanisms of this pathological flow-induced high-risk plaque remain largely unknown mainly because of the lack of such high-risk plaque animal models, hence there is an urgent need to correct this gap. Our long-term goal is to determine how the high-risk plaque is formed by pathological flow. We found that within 14 days after inducing LATS (large tumor suppressor homolog) 1/2 deletion in tamoxifen-inducible endothelial cell (EC) specific LATS1homo/LATS2homoknock-out mice (LATS1homo/LATS2homo-EKO), all mice (28/28) died of severe systemic edema, accompanied by massive EC apoptosis. Next we generated EC- specific LATS1het/LATS2homo-EKO hypercholesteremic (H-chol) mice; we detected a plaque erosion-like lesions at h- and d-flow areas in the aortic arch and carotids, which revealed strong fibrin/fibrinogen positive organized thrombus formation without a large necrotic core. We observed significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression, and 4) inflammation, which are collectively referred to as the ?pro-thrombotic phenotype?. We hypothesize that pathological flow-induced LATS1/2 degradation promotes EC damage-mediated thrombus formation and luminal re-endothelialization in concert. This cycle of EC damage-thrombus-re-endothelialization results in largethrombus formation and plaque erosion. Pathological flow-induced MAGI1 S741 phosphorylation by PKC?, and the subsequent LATS1/2-TERF2IP-MKRN1 complex formation are essential for LATS1/2 degradation. To test ths hypothesis, we propose the following 3 aims. In aim 1, we will characterize the role of excess EC proliferation, senescence/apoptosis, tissue factor (TF) expression, and inflammation in the formation of plaque erosion-like lesions and intraplaque hemorrhage in EC specific Lats1het/Lats2homo knock-out (Lats1het/Lats2homo-EKO) mice under H-chol. In aim 2, we will determine the crucial role of phosphorylation dependent TERF2IP and MKRN1 Ub E3 ligase binding in pathological flow-induced LATS1/2 destabilization and the consequent pro-thrombotic phenotype, and in aim 3 we will Investigate the role of PKC?-induced MAGI1 S741 phosphorylation in LATS1/2-mediated TERF2IP S205 phosphorylation and LATS1/2 degradation. The proposed work is expected to establish the link between pathological flow and pro-thrombotic phenotype, by which high-risk plaques are formed. The proposed study is innovative because it will propose a new concept how pathological flow affects the endothelium and induce the formation of vulnerable plaques of erosion and will provide insights into new signaling cascades and molecules responsible for this pathology. The proposed research may also provide means to predict and prevent high-risk plaque formation.